Recent evidence indicates that the axonal cytoskeleton plays important roles in axonal growth and in the production of certain forms of axonal pathology. Tubulin, one of the major proteins of the cytoskeleton, appears to play important roles in the maintenance of the intact axon and the outgrowth of the regenerating axon. Preliminary studies indicate that the axon normally contains significant quantities of unassembled tubulin, which appear to undergo polymerization as it is transported distally in the axon. In these studies, tubulin in motor axons of the sciatic nerve was labeled by axonal transport after the intraspinal administration of [35S] methionine. Unassembled and polymerized tubulin in the nerve was fractionated using the method of Pipeleers et al. [1] and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel fluorography. Levels of radioactivity in the unasembled and polymerized forms of axonal tubulin were measured using liquid scintillation spectroscopy. The proposed studies will use these techniques to investigate the role of unassembled tubulin in the growth and maintenance of the intact axon and in the pathogenesis of axonal disease. The axonal transport and state of polymerization of tubulin will be compared in three settings: 1) in normal axons; 2) during regenerative outgrowth; and 3) during acrylamide intoxication, in which there is a high degree of suspicion that the resulting impairment of outgrowth is related to alterations in axonal tubulin. First, these studies will reveal the behavior of unassembled and polymerized tubulin in normal axons. Second, they will test the hypothesis that the local polymerization of tubulin in regenerating sprouts is a necessary step in axonal outgrowth. Third, the behavior of tubulin will be examined in the presence of acrylamide, and agent which severely impairs regenerative outgrowth. If tubulin is altered by acrylamide, then it is likely that these changes are also involved in the pathogenesis of the dying-back pattern of axonal degeneration observed in intact axons during acrylamide intoxication. In summary, these studies should provide new insights into the role of tubulin in the normal maintenance of the healthy axon, the outgrowth of the regenerating axon, and the pathogenesis of at least one form of axonal disease.